Breast cancer is the second leading cause of cancer death among American women with over 150,000 new cases diagnosed and 50,000 deaths per year. Although a number of epidemiologic studies have suggested a relationship between dietary carotenoids and breast cancer risk, the molecular mechanisms contributing to the chemopreventive effects of carotenoids remain unknown. The studies in this proposal are aimed at identifying molecular targets of carotenoid action in mammary cells that may serve as suitable intermediate endpoints for dietary intervention and chemoprevention studies. We postulate that carotenoids and retinoids exert their chemopreventative effects by modulating autocrine and paracrine mammary growth regulatory pathways. We will utilize primary low passage cultures of normal human mammary epithelium and stromal cells, as well as mammary carcinoma cells, to examine the effects of carotenoids and retinoids on the expression of genes involved in the autocrine and paracrine regulation of mammary growth and differentiation. We will first investigate the effects of carotenoids and retinoids on the expression of gap junction proteins, connexin 26 and 43, and subsequent effects on homotypic and heterotypic cell-cell communication. By gene transfection we will determine the role of gap junction proteins in mediating stromal epithelial interaction. We will then determine whether carotenoids and retinoids modulate mammary growth through regulation of steroid hormone receptors including estrogen, progesterone and retinoic acid receptors, or through modulation of polypeptide growth factors such as TGFbeta and mammastatin. By examining effects on both mammary stroma and epithelium we will determine the role of stromal epithelial interactions in mediating the effects of carotenoids. These in vitro studies will help to elucidate the molecular mechanisms by which carotenoids and retinoids effect mammary growth and differentiation. Furthermore, they will identify suitable molecular markers to be used as intermediate endpoints in the dietary intervention studies proposed in an accompanying proposal. In these studies the effects of dietary carotenoids on the expression of genes identified in this proposal will be assessed in biopsy specimens from normal women, women with premalignant lesions, and women with breast cancer. In total, these interactive projects should provide important information on the role of dietary carotenoids in the prevention of breast cancer.